JP2021185877A - Cell culture container, cell culture method and evaluation method of cell growth state - Google Patents

Abstract

To provide a cell culture container capable of reducing a use amount of a culture component in a culture medium, and supplying properly, a medium according to a growth state of a cell in a closed system.SOLUTION: A cell culture container comprises: a culture chamber for culturing a cell; a storage chamber for storing fluid; a substance exchange film for partitioning the culture chamber and the storage chamber, and selectively transmitting a prescribed substance without transmitting the cell; and a gas exchange film for partitioning the culture chamber or the storage chamber from an external part. The culture chamber and the storage chamber have respectively, an opening part into which fluid can enter and from which the fluid can exit.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell culture vessel, a cell culture method for culturing cells using the cell culture vessel, and a cell growth state evaluation method for evaluating a cell growth state by the cell culture method.

Development of cell manufacturing technology using an automatic culture device for cell culture for medical and pharmaceutical applications is in progress. The automatic culture device is easier to mass-culture by expanding the culture field than the conventional culture by human technique, and the risk of contamination during cell culture can be significantly reduced by constructing a closed system. Is advantageous.

Various such automatic culture devices and related techniques have been developed. For example, Patent Document 1 proposes a suspension culture type automatic culture device in a culture tank. Further, Patent Document 2 proposes a back-type culture container applicable to an automatic culture apparatus.

Further, Patent Document 3 describes a cell culture apparatus including a culture medium image chamber separated by a dialysis membrane and a cell culture container having a culture image chamber. In the cell culture vessel of this cell culture device, the culture medium is located above the culture medium, and the cell suspension and the medium are separately supplied to the culture medium and the medium, so that the cells can be supplied without changing the medium. Long-term culture is possible.

Japanese Unexamined Patent Publication No. 2015-216880 Japanese Unexamined Patent Publication No. 2009-136156 Japanese Unexamined Patent Publication No. 61-108373

In general, the medium used for cell culture includes not only the nutritional components consumed by cells during growth, but also protein components such as components that stabilize the medium environment and bioactive substance components that affect the growth and differentiation of cells in a small amount. Contains the culture components of. Many of these culture components are derived from living organisms, and are present in the medium due to the addition of serum, purified protein, or the like, or the secretion of cells themselves during culture.

During the culture, the cells consume the nutrients contained in the culture medium and excrete waste products. Therefore, since the deterioration of the medium progresses as the cells grow, it is necessary to periodically replace the used culture medium with a fresh culture medium in general cell culture. On the other hand, at the time of this medium exchange, the culture components remaining in the culture medium are unnecessarily discarded. Therefore, there is a problem that the conventional cell culture method cannot efficiently use the culture components. In particular, in automatic culture equipment for industrial use, the amount of medium consumed due to the expansion of the culture field is increasing, so that the loss of culture components due to this problem cannot be ignored.

On the other hand, in the cell culture vessel of the cell culture apparatus described in Patent Document 3, cells are cultured by adding a culture medium containing serum to the culture medium and adding a nutrient medium containing no serum to the culture medium. is doing. However, this cell culture vessel is configured for long-term cell culture without changing the medium, and is applicable to the operation of appropriately supplying the medium according to the growth state of the cells and the operation of culturing the cells in a closed system. Can not do it.

Furthermore, in recent years, there has been a need for mass culture of cells by three-dimensional culture and production by culture of three-dimensional tissues expressing high functionality. In these cultures, the cell density is significantly increased as compared with the conventional planar culture, and therefore the deterioration rate of the medium is increased accordingly. Therefore, in order to store and supply a large amount of medium, a structure that is not restricted by the medium volume and a structure that can quickly and frequently supply fresh medium are required.

The present invention has been made in view of the above problems, and is a cell culture capable of reducing the amount of culture components used in a culture medium and appropriately supplying a medium according to the growth state of cells in a closed system. An object of the present invention is to provide a container, a cell culture method for culturing cells using the cell culture container, and a cell growth state evaluation method for evaluating the cell growth state by the cell culture method.

In order to solve the above problems, the cell culture vessel of the present invention comprises a culture chamber for culturing cells, a storage chamber for storing fluid, the culture chamber and the storage chamber, and is predetermined without permeating the cells. A substance exchange membrane that selectively permeates the above-mentioned substances and a gas exchange membrane that separates the outside from the above-mentioned culture chamber or the above-mentioned storage chamber, and the above-mentioned culture chamber and the above-mentioned storage chamber each have an opening through which fluid can flow in and out. It is characterized by having.

Further, in order to solve the above-mentioned problems, the cell culture method of the present invention is a cell culture method in which the above-mentioned cells are cultured in the above-mentioned culture chamber using the above-mentioned cell culture vessel, and the above-mentioned culture chamber is passed through the above-mentioned opening. And the above-mentioned storage chamber is supplied with a medium having different components or component concentrations.

Further, in order to solve the above problems, the method for evaluating the cell growth state of the present invention is a method for evaluating the cell growth state in which the cell growth state is evaluated in the process of culturing the cells by the cell culture method. Then, the difference in glucose concentration between the medium supplied to the storage chamber and the medium discharged from the storage chamber is measured, the glucose consumption rate is calculated from the difference in glucose concentration, and the culture is obtained from the glucose consumption rate. It is characterized by estimating the number of cells in the room.

According to the present invention, the amount of the culture component used in the culture medium can be reduced, and the medium can be appropriately supplied according to the growth state of the cells in a closed system.

Issues, configurations, and effects of the present invention other than those described above will be clarified by the following description of embodiments for carrying out the invention.

It is a schematic sectional drawing which shows the main part of the example of the cell culture container of an embodiment. FIG. 3 is a schematic cross-sectional view taken along the line AA shown in FIG. It is a figure which shows the main part of the modification of the cell culture container of an embodiment, and is the schematic cross-sectional view which shows the cross section corresponding to the cross section shown in FIG. It is a schematic cross-sectional view which shows the main part of the other modification of the cell culture vessel of an embodiment. (A) is a graph showing the time course of the glucose concentration in the outside (external solution) and the inside (internal solution) of the bag-shaped dialysis membrane, and (b) is the outside (external solution) and the inside of the bag-shaped dialysis membrane. It is a graph which shows the time-dependent change of the lactic acid concentration in (internal solution). It is a schematic sectional drawing which shows the cell culture container designed in Example 1. FIG.

Hereinafter, embodiments relating to the cell culture vessel, the cell culture method, and the method for evaluating the cell growth state of the present invention will be described.

First, an example of the cell culture vessel of the embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is a schematic cross-sectional view showing a main part of an example of a cell culture vessel of an embodiment. FIG. 2 is a schematic cross-sectional view taken along the line AA shown in FIG.

As shown in FIG. 1, the cell culture vessel 1 of this example includes a housing 5, and has a culture chamber 10 for culturing cells C and a storage chamber 20 for storing fluid inside the housing 5. .. The cell culture vessel 1 has a substance exchange membrane 30 that partitions the culture chamber 10 and the storage chamber 20 and selectively permeates a predetermined substance, and a gas exchange membrane 40 that separates the culture chamber 10 and the storage chamber 20 from the outside air, respectively. Is further equipped. The side surface side of the culture chamber 10 is separated from the outside air by the housing 5, the lower surface side is separated from the outside air by the gas exchange membrane 40, and the upper surface side is separated from the storage chamber 20 by the substance exchange membrane 30. The side surface side of the storage chamber 20 is separated from the outside air by the housing 5, the upper surface side is separated from the outside air by the gas exchange membrane 40, and the lower surface side is separated from the culture chamber 10 by the substance exchange membrane 30. The substance exchange membrane 30 allows the nutrient components contained in the nutrient medium supplied to the storage chamber 20 and low molecular weight components such as waste products generated in the culture to permeate the culture medium 10 and becomes the culture medium supplied to the culture chamber 10. It is a membrane having selective permeability that does not permeate high molecular weight protein components such as contained physiologically active substance components. The gas exchange membrane 40 is a membrane that permeates oxygen and does not permeate the medium.

Ports 51 and 52 (openings through which fluid can flow in and out) are provided at two opposite locations on the side surface of the culture chamber 10, and opening / closing mechanisms (not shown) are provided at each of the ports 51 and 52. ing. Ports 61 and 62 (openings capable of inflowing and discharging fluid) are provided at two opposite locations on the side surface of the storage chamber 20, and opening / closing mechanisms (not shown) are provided at each of the ports 61 and 62. ..

Following this, as an example of the cell culture method of the embodiment, a cell culture method of culturing cells C in the culture chamber 10 using the cell culture container 1 shown in FIG. 1 will be described.

In the cell culture method of this example, the culture scaffold 80 (a structure capable of adhering or embedding cells) and the cell C adhering to the culture scaffold 80 are introduced into the culture chamber 10 of the cell culture container 1 in advance. Further, by connecting a pipe (not shown) to the port 51 of the culture chamber 10 and connecting the side of the pipe opposite to the port 51 to a culture medium supply source (not shown) or the like, an inflow path is constructed and culture is performed. An outflow path is constructed by connecting a pipe (not shown) to the port 52 of the chamber 10 and connecting the side of the pipe opposite to the port 52 to a recovery tank or the like. Further, by connecting a pipe (not shown) to the port 61 of the storage chamber 20 and connecting the side of the pipe opposite to the port 61 to a nutrient medium supply source (not shown) or the like, an inflow path is constructed and storage is performed. An outflow path is constructed by connecting a pipe (not shown) to the port 62 of the chamber 20 and connecting the side of the pipe opposite to the port 62 to a storage tank or the like. Then, a sampling port S1 or a component analysis sensor S2 is installed in each of the inflow path and the outflow path connected to the storage chamber 20. As a result, a closed cell culture apparatus composed of a cell culture vessel 1, a culture medium supply source and a nutrient medium supply source, an inflow channel and an outflow channel, a recovery tank and a storage tank, and a sampling port S1 or a component analysis sensor S2. To build. In this cell culture apparatus, cells can be continuously cultured by perfusing a medium, gas, or the like into a storage chamber 20 or a culture chamber 10 via an inflow channel and an outflow channel.

Subsequently, the cell culture vessel 1 is placed in an incubator (not shown), and the cells are cultured in the culture chamber 10 of the cell culture vessel 1. At this time, first, by opening the ports 61 and 62 of the storage chamber 20 of the cell culture vessel 1 by the opening / closing mechanism, the nutrient medium supply source is pushed out from the storage chamber 20 to the outflow passage through the port 62. After filling the storage chamber 20 with the fresh nutrient medium by supplying the storage chamber 20 with the fresh nutrient medium via the port 61, the ports 61 and 62 are closed by the opening / closing mechanism. Next, by opening the ports 51 and 52 of the culture chamber 10 by an opening / closing mechanism, air is pushed out from the culture chamber 10 to the outflow channel through the port 52, and the culture medium supply source passes through the culture chamber 10 through the port 51. After filling the culture chamber 10 with the culture medium by supplying a liquid culture medium to the culture medium, the ports 51 and 52 are closed by an opening / closing mechanism. Next, by continuously opening the ports 61 and 62 of the storage chamber 20 by the opening / closing mechanism, a fresh liquid nutrient medium containing a nutrient component is continuously supplied from the nutrient medium supply source to the storage chamber 20 via the port 61. The used nutrient medium containing the waste products generated by the culture is continuously discharged from the storage chamber 20 through the port 62 to the recovery tank. As a result, the nutritional components contained in the nutrient medium supplied to the storage chamber 20 are supplied to the culture chamber 10 by permeating the substance exchange membrane 30, and the waste products generated by the culture permeate the substance exchange membrane 30 into the culture chamber 10. By doing so, it moves to the storage chamber 20. On the other hand, the protein component contained in the culture medium supplied to the culture chamber 10 is retained in the culture chamber 10. As a result of such dialysis, in a closed cell culture apparatus, continuous supply of fresh nutrient medium from the culture medium source to the reservoir 20 and continuous supply of used nutrient medium from the reservoir 20 to the recovery tank. By performing such discharge, deterioration of the culture medium in the culture chamber 10 can be prevented, and the protein component contained in the culture medium can be retained in the culture chamber 10. Therefore, in a closed cell culture apparatus, cells can be cultured in the culture chamber 10 without exchanging the culture medium in the culture chamber 10. Therefore, the amount of the protein component used in the culture medium can be reduced, the production cost of the cells C can be suppressed, and the medium can be supplied in a closed system.

Following this, as an example of the method for evaluating the cell growth state of the embodiment, the method for evaluating the cell growth state for evaluating the growth state of the cell C will be described in the above example of the cell culture method.

In the method for evaluating the cell growth state of this example, sampling installed in each of the inflow channel and the outflow channel of the storage chamber 20 in the process of culturing the cells C in the culture chamber 10 of the cell culture vessel 1 by the cell culture method of the above example. After measuring the difference in glucose concentration between the nutrient medium supplied to the storage chamber 20 and the nutrient medium discharged from the storage chamber 20 using the port S1 or the component analysis sensor S2, the glucose consumption rate is calculated from the difference in glucose concentration. Calculate and estimate the number of cells in the culture chamber 10 from the glucose consumption rate. Further, after measuring the lactic acid concentration of the nutrient medium discharged from the storage chamber 20, the lactic acid production rate is calculated from the lactic acid concentration, and the metabolic behavior of the cells in the culture chamber 10 is estimated from the glucose consumption rate and the lactic acid production rate. Then, in the cell culture method of the above example, the growth state of cells is evaluated from the number of cells in the culture chamber 10 and the metabolic behavior of the cells estimated in this way, and based on the evaluation result, the storage chamber 20 or the culture chamber is used. The components of the nutrient medium supplied to 10 can be adjusted. Therefore, the cell culture vessel 1 can be applied to a cell culture device that appropriately supplies a medium according to the growth state of cells in a closed system.

Therefore, the cell culture vessel of the embodiment can reduce the amount of the culture component used in the culture medium and suppress the cell production cost as in the above example, and is a closed system depending on the cell growth state. The medium can be supplied appropriately. Further, according to the cell culture method of the embodiment, by culturing the cells in the culture chamber using the cell culture vessel of the embodiment, the amount of the culture component used in the culture medium is reduced and the cell production cost is suppressed. It is possible to supply a medium appropriately according to the growth state of cells in a closed system. Further, according to the method for evaluating the cell growth state of the embodiment, in the process of culturing the cells by the cell culture method of the embodiment, the number of cells in the culture chamber and the metabolic behavior of the cells are estimated, and the cell growth state is evaluated. be able to.

Hereinafter, the configuration of the cell culture vessel of the embodiment, the cell culture method of the embodiment, and the evaluation method of the cell growth state will be described in detail.

1. 1. Substance exchange membrane The substance exchange membrane is a membrane that partitions the culture chamber and the storage chamber and selectively permeates a predetermined substance. The substance exchange membrane is not particularly limited as long as it is a membrane having selective permeability that selectively permeates a predetermined substance such as gas or solution, but the nutritional components contained in the nutrient medium and waste products generated in culture are used. Those having selective permeability that permeates low molecular weight components and does not permeate target high molecular weight protein components such as physiologically active substance components contained in the culture medium are preferable. By using the substance exchange membrane having such selective permeability, the nutrient components contained in the nutrient medium and the waste products generated in the culture can be exchanged between the storage chamber and the culture chamber, and are contained in the culture medium. The protein component can be retained in the culture chamber.

The substance exchange membrane needs to permeate low molecular weight components such as nutritional components and waste products as described above, and does not need to permeate the target high molecular weight protein components. Examples of the nutritional component include glucose (molecular weight: 180.96) and lactic acid (molecular weight: 90.08), and examples of the target high molecular weight protein component include bovine serum, which is a protein stabilizer. Examples thereof include albumin (molecular weight: 66000) and FGF2 (molecular weight: 17400) which is a growth factor. Therefore, the fractional molecular weight (MWCO) of the substance exchange membrane is not particularly limited as long as the substance exchange membrane is a membrane having selective permeability as described above, but is, for example, in the range of 500 or more and 60,000 or less. It is preferable, and above all, the range of 1000 or more and 15,000 or less is preferable. This is because the target high molecular weight protein component such as these can be retained in the culture chamber. Even when a protein component other than bovine serum albumin and FGF2 is targeted, the target protein component can be retained in the culture chamber by using a substance exchange membrane having an appropriate molecular weight cut-off. Here, the "molecular weight cut-off" refers to the molecular weight of the spherical solute when a plurality of types of low-concentration spherical solutes are permeated through the substance exchange membrane and the inhibition rate is 90% or more.

The substance exchange membrane is not particularly limited as long as it has the above-mentioned selective permeability, and is, for example, a semipermeable membrane or the like. Examples of the semipermeable membrane include a cellulosic membrane (for example, a regenerated cellulose membrane, a surfaced modified regenerated cellulose membrane, a cellulose acetate, etc.) and a synthetic polymer membrane (for example, polyacrylonitril, polymethylmethacrylate, ethylenevinyl, etc.). Alcohol copolymers, polysulfones, polyamides, polyester polymer alloys, fluororesins, etc.) and the like can be mentioned. Further, the substance exchange membrane is preferably a cell non-adhesive membrane.

2. 2. Gas exchange membrane A gas exchange membrane is a membrane that allows oxygen to pass through. Since the cell density is high in the three-dimensional culture of cells, the lack of oxygen required for cell growth becomes an issue. Therefore, by installing a gas exchange membrane in the vicinity of the culture scaffold in the culture chamber, oxygen can be efficiently supplied from the outside air. As the gas exchange membrane, a cell non-adhesive membrane is preferable. This is because the use of a cell non-adhesive membrane can prevent cells from detaching from the culture scaffold to the bottom surface and migrating.

The gas exchange membrane is not particularly limited as long as it is a membrane that allows oxygen to pass through, but for example, it is mainly composed of any one of silicone, polytrimethylsilylpropine, fluoroacrylic resin, fluororesin, polymethylpentene, and natural rubber. And the like are preferable.

3. 3. Cell culture container The cell culture container divides a culture chamber for culturing cells, a storage chamber for storing fluid, the culture chamber and the storage chamber, and selectively permeates a predetermined substance without permeating the cells. It comprises a substance exchange membrane and a gas exchange membrane that separates the outside from the culture chamber or the storage chamber, and each of the culture chamber and the storage chamber has an opening through which fluid can flow in and out.

The housing of the cell culture vessel is not particularly limited, but for example, one having (a) biocompatibility, (b) sterilizable, (c) corrosion resistance, and (d) moldable. Among them, glass, resins such as polystyrene, polycarbonate and polyethylene terephthalate used in commercially available culture containers, medical resins such as MED610, stainless steel such as SUS316, and titanium are preferable.

The culture chamber and the storage chamber each have an opening through which a fluid such as a medium can flow in and out. Fluids such as media and gas and cells can flow in and out through these ports. The openings of the culture chamber may be at least two openings that allow the fluid to flow in and out, as in ports 51 and 52 shown in FIG. 1, and three or more openings that allow the fluid to flow in and out. It may be an opening. Similarly, the openings of the storage chamber may be at least two openings that allow the fluid to flow in and out, as in ports 61 and 62 shown in FIG. 1, and the fluid can flow in and out 3 It may be one or more openings. The storage chamber or culture chamber may further include an opening / closing mechanism provided at each opening. By the opening / closing mechanism of the opening, it is possible to set the supply of the fluid to the storage chamber or the culture chamber and the discharge of the fluid from the storage chamber or the culture chamber continuously or sequentially. Since the substance exchange between the storage chamber and the culture chamber via the substance exchange membrane occurs at the interface between the liquids, if air remains on the surface of the substance exchange membrane, the efficiency of the substance exchange via the substance exchange membrane decreases. Sometimes. Therefore, by providing at least two openings in each of the storage chamber and the culture chamber, air can be pushed out when the liquid of the medium or the liquid containing cells flows into each chamber, and the liquid of the medium or the cells can be extruded. Since each chamber can be easily filled with the medium without leaving air in the flow path of the liquid or the like containing the above (for example, the surface of the substance exchange membrane), it is possible to suppress a decrease in the efficiency of the substance exchange. On the other hand, when only one opening is provided in each of the storage chamber and the culture chamber, each chamber is not left with air when the liquid of the medium or the liquid containing cells is allowed to flow into each chamber. Difficult to fill with medium.

Here, FIG. 3 is a diagram showing a main part of a modified example of the cell culture container of the embodiment, and is a schematic cross-sectional view showing a cross section corresponding to the cross section shown in FIG. As shown in FIG. 3, the storage chamber 20 of the cell culture vessel 1 of this example may have a port 63 (opening) for bleeding air in addition to the ports 61 and 62 (opening). Similarly, the culture chamber 10 may have a port 53 (opening) for venting air in addition to the ports 51 and 52 (opening).

The storage chamber or culture chamber may have a plurality of openings that allow the fluid to flow in from the outside and a plurality of openings that allow the fluid to flow out to the outside. In this case, the storage chamber or the culture chamber may further include an opening / closing mechanism provided at each opening. As a result, different media supply sources are prepared, and by connecting the different culture medium supply sources to the plurality of openings of the storage chamber or the culture chamber, the type of medium to be introduced into the storage chamber or the culture chamber can be selected. It can be continuously switched, and the components of the medium to be introduced can be adjusted by inflowing and mixing a plurality of types of media into the storage chamber or the culture chamber. Of course, a flow path may be constructed by connecting to one opening of the storage chamber or the culture chamber, branching the opposite side of the opening, and connecting to each of a plurality of culture medium supply sources by piping or the like. In this case as well, it is possible to continuously switch the type of medium and adjust the components of the medium.

Here, FIG. 4 is a schematic cross-sectional view showing a main part of another modification of the cell culture vessel of the embodiment. As shown in FIG. 4, the cell culture container 1 of this example has a culture chamber 10 for culturing cells C and a first storage chamber 21 and a second storage chamber 22 for storing fluid, respectively, inside the housing 5. It may have a stepped structure. In this case, the cell culture vessel 1 partitions the culture chamber 10 and the first storage chamber 21, and has a first substance exchange membrane 31 that selectively permeates a predetermined substance, and the culture chamber 10 and the second storage chamber 22. A second substance exchange membrane 32 that selectively permeates a predetermined substance, a first gas exchange membrane 41 that separates the first storage chamber 21 from the outside air, and a second gas that separates the second storage chamber 22 from the outside air. It further comprises an exchange membrane 42. The side surface side of the culture chamber 10 is separated from the outside air by the housing 5, the upper surface side is separated from the first storage chamber 21 by the first substance exchange membrane 31, and the lower surface side is separated from the second storage chamber 22 by the second substance exchange membrane 32. Has been done. The side surface side of the first storage chamber 21 is separated from the outside air by the housing 5, the upper surface side is separated from the outside air by the first gas exchange membrane 41, and the lower surface side is separated from the culture chamber 10 by the first substance exchange membrane 31. The side surface side of the second storage chamber 22 is separated from the outside air by the housing 5, the upper surface side is separated from the outside air by the second substance exchange membrane 32, and the lower surface side is separated from the outside air by the second gas exchange membrane 42. Ports 51 and 52 (openings through which fluid can flow in and out) are provided at two opposite positions on the side surface of the culture chamber 10, respectively. Ports 61 and 62 (openings through which fluid can flow in and out) are provided at two opposite locations on the side surface of the first storage chamber 21, respectively. Ports 61 and 62 (openings through which fluid can flow in and out) are provided at two opposite locations on the side surface of the second storage chamber 22, respectively.

When the cell culture vessel 1 has such a three-stage structure, the first storage chamber 21 and the second storage chamber 22 enter the culture chamber 10 via the first substance exchange membrane 31 and the second substance exchange membrane 32, respectively. Since they are adjacent to each other, the effective area for substance exchange increases, and substance exchange becomes possible more efficiently. For example, when the first substance exchange film 31 and the second substance exchange film 32 have the same selective permeability of permeating low molecular weight components in the same range and not permeating high molecular weight protein components in the same range. When the same nutrient medium is supplied to both the first storage chamber 21 and the second storage chamber 22, the nutritional components can be supplied to the culture chamber 10 more efficiently, and the waste products can be more efficiently supplied from the culture chamber 10. Can be discharged as a target. On the other hand, when the first substance exchange membrane 31 and the second substance exchange membrane 32 have different selective permeability of permeating components having different molecular weights, a substance that selectively exchanges a plurality of types of substances. Can be exchanged. For example, when the first substance exchange membrane 31 is a dialysis membrane that permeates low molecular weight components such as nutrients and waste products and the second substance exchange membrane 32 is a gas exchange membrane, the first storage chamber 21 is used for nutrition. By supplying a medium and supplying a gas having an adjusted oxygen concentration to the second storage chamber 22, oxygen is efficiently supplied to the culture chamber 10, and nutrient components and waste products are supplied to the first storage chamber 21 and the culture chamber. It can be exchanged between 10.

4. Cell culture method and evaluation method of cell growth state The cell culture method is a cell culture method in which the cells are cultured in the culture chamber using the cell culture vessel, and the culture chamber and the storage are provided through the opening. It is characterized in that a medium having different components or component concentrations is supplied to the chamber.

The cells to be cultured in the culture chamber may be either adherent cells or floating cells, may be one type of cells, or may be a plurality of types of cells. Further, the cells to be cultured in the culture chamber are not limited to a single cell existing in a dispersed manner, and may be a spheroid or a tissue containing cells and extracellular matrix.

When the cells to be cultured in the culture chamber are adherent cells, it is preferable to introduce a culture scaffold into the culture chamber. A culture scaffold is a structure capable of adhering or embedding cells. The material of the culture scaffold is not particularly limited as long as it is a biocompatible material capable of adhering or embedding cells, but for example, a porous body or gel composed of silicone, resin, glass, polysaccharide, protein or the like. , Films, discs, beads and the like.

Examples of the method of introducing the cells or the culture scaffold into the culture chamber include a method of flowing the cells or the culture scaffold into the culture chamber through openings such as ports 51 and 52 shown in FIG. When the culture chamber is an assembly type, the cells or the culture scaffold can be introduced into the culture chamber by assembling the culture chamber after introducing the cells or the culture scaffold into the culture chamber before the assembly in advance.

The medium to be introduced into the culture chamber is not particularly limited as long as it has a different component or component concentration from the medium supplied to the storage chamber, and for example, a culture medium containing a culture component such as a protein component necessary for cell culture may be used. Can be mentioned. In cell culture, in addition to nutritional components that are subject to assimilation or catabolism, for example, protein components such as physiologically active substance components that affect cell proliferation and differentiation in small amounts and components that stabilize the medium environment. Culture components are needed. Examples of the bioactive substance component include growth factors such as cytokines and hormones, pharmaceuticals and the like. Culture components such as protein components necessary for culturing these cells can be contained in the medium by adding serum, purified protein, or the like, or by secreting the cells themselves during culture.

The medium supplied to the storage chamber is not particularly limited as long as it has a different component or component concentration from the medium supplied to the culture chamber, and examples thereof include a nutritional medium containing a nutritional component consumed by cells during growth. Among them, a nutrient medium or the like having a protein component concentration of 1% or less is preferable. Examples of the nutritional component include sugars such as glucose, amino acids, lipids, peptides, nucleic acids and the like. As the nutrient medium, for example, a basal medium containing amino acids, vitamins, inorganic salts, glucose and the like is preferable.

As a cell culture method, the medium is continuously or sequentially supplied to the storage chamber through the opening, and the medium is continuously or sequentially discharged from the storage chamber through the opening. , The method of culturing the cells in the culture chamber is preferable. This allows the cells to be continuously cultured. Among these methods, a method of culturing the cells in the culture chamber in a state where the sampling port or the component analysis sensor is installed in the opening of the storage chamber or the flow path connected to the opening is preferable. In particular, using the sampling port or the component analysis sensor, glucose, lactic acid, ammonia, glutamine, glutamic acid, oxygen, and carbon dioxide contained in the medium (the carbon dioxide is carbonic acid, carbonate ion, or hydrogen carbonate ion). A method of culturing the cells in the culture chamber while continuously or sequentially measuring the concentration of at least one component (including) is preferable. Thereby, the cell growth state can be evaluated by analyzing the components of the medium supplied to the storage chamber and the medium discharged from the storage chamber and estimating the number of cells and the metabolism of the cells in the culture chamber. Then, based on the evaluation result of the cell growth state, the method of supplying the medium to the storage chamber or the culture chamber can be controlled by feedback control.

Hereinafter, the grounds for obtaining the effect by the cell culture method of the present invention will be theoretically described. In the following, the results of verifying the applicability of the cell culture method of the present invention to high-density culture of hMSC (human mesenchymal stem cells), which is one of the cells that have received the most attention in cell therapy, will be described in detail. The rationale will be explained theoretically. In the verification of this applicability, since the cell density in the confluent state in the general planar culture of hMSC is ^{about 1 × 10 5} cells / cm ² , the achievement target of the cell density is 1 × 10 ^{6 which is 10 times that.} It was set to cells / cm ² . This applicability was verified by using a commercially available dialysis membrane Spectra / Por 7 (MWCO: 10 kDa) as a substance exchange membrane and investigating the dialysis characteristics of the dialysis membrane for glucose and lactic acid.

In order to investigate the dialysis characteristics of the dialysis membrane for glucose and lactic acid, first, the lactic acid concentration (solute concentration) is 2. Fill with an internal solution of 0 g / L, fill the outside of the bag-shaped dialysis membrane with an external solution having a glucose concentration (solute concentration) of 4.5 g / L, and fill the outside (external solution) and inside (internal solution) of the bag-shaped dialysis membrane. The time course of glucose concentration and lactic acid concentration in dialysis was measured. Here, FIG. 5A is a graph showing changes over time in glucose concentration in the external (external solution) and internal (internal solution) of the bag-shaped dialysis membrane, and FIG. 5B is a graph of the bag-shaped dialysis membrane. It is a graph which shows the time-dependent change of the lactic acid concentration in the external (external solution) and the internal (internal solution).

In general, mass transfer through the dialysis membrane is due to diffusion. Therefore, in two kinds of solutions that are in contact with each other across the dialysis membrane, the transfer rate of the solute through the dialysis membrane between one solution and the other solution d (C ₁ V ₁ ) / dt [here, C ₁ is the solute concentration of the one solution, and V ₁ is the volume of the one solution] is the difference in the solute concentration between the one solution and the other solution ΔC [where ΔC = C ₁ −. C ₀ , where C ₀ is the solute concentration of the other solution], _{proportional to the area of the dialysis membrane Smembrane} , and the permeation coefficient k peculiar to the dialysis membrane, and shall be expressed by the differential equation of the following equation (1). Can be done.

On the other hand, in the time course of the glucose concentration shown in FIG. 5 (a), the glucose concentration (solute) in the outer (external solution) and the inner (internal solution) of the bag-shaped dialysis membrane in contact with the bag-shaped dialysis membrane. The difference ΔC _{gluc (} concentration) indicates an exponential time dependence. Similarly, in the time course of the lactic acid concentration shown in FIG. 5 (b), the lactic acid concentration (solute concentration) in the outer (external solution) and the inner (internal solution) of the bag-shaped dialysis membrane in contact with the bag-shaped dialysis membrane. ) Difference ΔC _lac indicates an exponential time dependence. Therefore, when focusing on the inside (internal solution) of the bag-shaped dialysis membrane, the movement speed d of glucose (solute) via the dialysis membrane between the outside (external solution) and the inside (internal solution) of the bag-shaped dialysis membrane. (C _gluc V _inside ) / dt [Here, C _gluc is the glucose concentration _inside the bag-shaped dialysis membrane (internal solution), and V inside is the volume inside the bag-shaped dialysis membrane (internal solution)]. , The difference in glucose concentration between the outside (external solution) and the inside (internal solution) of the _{bag-shaped dialysis membrane is proportional to ΔC gluc} , and dialysis between the inside (internal solution) and the outside (external solution) of the bag-shaped dialysis membrane. Movement rate of lactic acid (solute) through the membrane d (C _lac V _inside ) / dt [Here, C _{lac is the lactic acid concentration inside} the sac-shaped dialysis membrane (internal solution), and V inside is the sac-shaped dialysis membrane. internal the volume (internal solution)] is, to be proportional to the difference [Delta] C _lac lactate concentration has been suggested in the external of the bag-like dialysis membrane (external solution) and internal (internal solution). The glucose concentration and the lactic acid concentration are substantially constant outside the bag-shaped dialysis membrane (external solution), and the volumes inside (internal solution) and outside (external solution) of the bag-shaped dialysis membrane are substantially constant. _{Therefore, the glucose (solute) transfer rate d (C gluc} V _inside ) / dt between the external (external solution) and the internal (internal solution) of the bag-shaped dialysis membrane is set to the glucose permeability coefficient of k _gluc. When it is expressed by the above equation (1), it can be expressed by approximating it by the following equation (2). Similarly, the transfer coefficient of lactic acid _(Clac V _inside ) / dt through the dialysis membrane between the inside (internal solution) and the outside (external solution) of the bag-shaped dialysis membrane is k. _{When the lac} is expressed by the above equation (1), it can be approximated by the following equation (3).

According to the above formula (2), the glucose permeation coefficient k _gluc is 2.2 _{by fitting the time dependence of the difference in glucose concentration ΔC gluc} with respect to the time course of the glucose concentration shown in FIG. It is calculated as × 10 ^-4 L / cm ^{2 / h.} Similarly, according to the above formula (3), the time course of the lactic acid concentration shown in FIG. 5 (b), by fitting the time dependence of the concentration difference [Delta] C _lac lactate concentration exponentially, permeability coefficient k _lac lactate Is calculated as 2.6 × 10 ^-4 L / cm ² / h.

Subsequently, the number of reached cells N _max is calculated using these permeability coefficients, and the reached cell density N _max / _Molare and the reached lactic acid concentration C _lac are estimated.

At the end of dialysis in the dialysis culture method, a dialysate containing a certain concentration of glucose and not containing lactic acid (corresponding to the above external solution) is depleted of glucose and dialysis with a culture solution containing a constant concentration of lactic acid (corresponding to the above internal solution). In the state of being in contact with each other across the membrane, the supply rate of glucose from the dialysate to the culture solution via the dialysate becomes balanced with the glucose consumption by hMSC, and the dialysate from the culture solution to the dialysate. It is considered that the discharge rate of dialysis via dialysis is in balance with the amount of lactic acid produced by hMSC. _Therefore, the glucose consumption rate of hMSC a _{r gluc,} lactate production rate of hMSC a _{r lac,} the glucose concentration of the dialysate _{C G,} when the arrival lactate concentration in the culture solution and _{C L,} the following formula (4) and (5) Is considered to hold.

Here, for simplicity, assume the conditions used a cylindrical cell chamber, i.e. the condition where the area _{S membrane} and cell contact area _{S culture} of the culture chamber of the dialysis membrane is the same, generally a glucose concentration _{C G} of the dialysate It is assumed that 1.0 g / L (low glucose medium) used in the culture is used. Further, hMSC is generally known to consume glucose at a rate of about ^{3 to 7 × 10-11 g / cell / h.} Therefore, the glucose consumption rate r _gluc of hMSC is overestimated to be 8 × 10 ^-11 g / cell / h. Furthermore, by assuming complete anaerobic respiration, the lactate production rate r _lac of hMSC is made the same as the glucose consumption rate r _{gluc of hMSC.} With the above settings, the reached cell density N _max / _Culture is estimated to be 2.8 × 10 ⁶ cells / cm ^{2 from the above formula (4).} This value exceeds the achievement target of cell density (1 × 10 ⁶ cells / cm ² ). Furthermore, reach lactate concentration _{C L} of the culture solution from the above equation (5) is estimated to 0.85 g / L. Therefore, since hMSC can be cultivated assuming that the lactic acid concentration of the culture solution is 1.0 g / L, hMSC is obtained by dialysis using the dialysis membrane Spectra / Por 7 (MWCO: 10 kDa) in this study. It is considered that lactic acid can be removed sufficiently for culturing.

Therefore, by applying the cell culture method of the present invention, it is considered that hMSC can be cultured at a density 10 times or more that of the conventional general planar culture. In the above-mentioned verification of the applicability of the cell culture method of the present invention, the applicability of hMSC to high-density culture was verified, but the application target of the cell culture method of the present invention is to high-density culture of hMSC. The cell culture method of the present invention is not limited, and can be applied to, for example, culture of other types of cells and culture for the purpose of differentiation or maintenance other than proliferation.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples. However, the present invention is not limited to the examples and reference examples listed below.

[Example 1]
The cell culture vessel of the present invention was designed. FIG. 6 is a schematic cross-sectional view showing the cell culture vessel designed in Example 1. Hereinafter, a production method based on the design of the cell culture vessel 1 shown in FIG. 6 will be specifically described.

When the housing 5 is manufactured in the production of the cell culture vessel 1, for example, a method using a medical resin (MED610) as a material and using a 3D printer, or a method of cutting stainless steel (SUS316) is used. Further, the upper part 5U of the housing which is the upper part of the housing 5 and the lower part 5L of the housing which is the lower part of the housing 5 are separately manufactured. A gas exchange film 40 is installed in the opening 5Ua provided on the upper surface of the upper portion 5U of the housing and the opening 5La provided on the lower surface of the lower portion 5L of the housing so as to close them. Then, the cell culture vessel 1 is manufactured by assembling the upper part 5U of the housing and the lower part 5L of the housing so as to sandwich the substance exchange membrane 30 between them.

In the method for producing the cell culture vessel 1, the cell culture vessel 1 can be assembled with the three-dimensional culture scaffold 80 introduced in advance in the culture chamber 10, which is the space inside the lower part 5L of the housing. Therefore, the culture scaffold 80 is not particularly limited as long as it has a size that fits in the culture chamber 10, and the shape and properties can be appropriately selected. Of course, when the culture scaffold 80 is a culture scaffold having high fluidity and dispersibility, a method of pouring the culture scaffold 80 into the culture chamber 10 as a fluid can be used by using the ports 51 and 52. In the cell culture vessel 1, the structure of the culture scaffold 80 introduced into the culture chamber 10 makes it possible to easily perform cell culture using the three-dimensional culture scaffold.

When the housing upper portion 5U is manufactured, the partition plate 24 is installed in the storage chamber 20, which is the space inside the housing upper portion 5U, along the direction perpendicular to the flow path direction. With such a structure of the upper portion 5U of the housing, when the medium flows into the storage chamber 20 from the port 61, the medium can be flowed in the vicinity of the substance exchange membrane 30 installed on the lower surface of the storage chamber 20. As a result, substances can be efficiently exchanged between the storage chamber 20 and the culture chamber 10.

When the lower part 5L of the housing is manufactured, the air reservoir 16 is formed on the upper side in the culture chamber 10. With this structure, even if air remains when the medium is supplied into the culture chamber 10, by storing the air in the air reservoir 16, it is possible to suppress a decrease in the efficiency of substance exchange due to bubbles attached to the substance exchange membrane 30. can do.

Hereinafter, an example of a cell culture method using the cell culture container 1 will be described.
In the cell culture method of this example, first, the upper 5U of the housing, the lower 5L of the housing, the gas exchange membrane 40, and the substance exchange membrane 30 are sterilized in advance before assembling the cell culture container 1. Then, the cell culture vessel 1 is assembled and the cells are seeded in a sterile environment.

Next, the tubes are connected to the ports 61 and 62 of the upper part 5U of the housing and the ports 51 and 52 of the lower part 5L of the housing, and sealed with a luer lock connector or a clamp. Further, the three-dimensional culture scaffold 80 is introduced into the culture chamber 10 of the lower part of the housing 5L from the opening 5Lb. At this time, the cells and the culture medium may be introduced into the culture chamber 10 at the same time.

Next, the cell culture vessel 1 is assembled by installing the substance exchange membrane 30 in the opening 5Lb of the housing lower part 5L and assembling the housing upper part 5U and the housing lower part 5L so as to sandwich the substance exchange membrane 30 between them. To make. The upper portion 5U of the housing and the lower portion 5L of the housing have dimensions such that the wall surfaces around the opening 5Ub and the opening 5Lb are in close contact with each other when the upper portion 5U of the housing and the lower portion 5L of the housing are assembled.

Next, after assembling the cell culture vessel 1, the ports 61 and 62 of the upper part 5U of the housing are opened, and a nutrient medium containing nutrients to be consumed during cell growth is introduced. After that, the ports 61 and 62 of the upper part 5U of the housing are closed.

Next, the ports 51 and 52 of the lower part 5L of the housing are opened, and a culture medium, which is a medium containing cells and protein components necessary for cell culture, is introduced. At that time, when the bubbles are in contact with the substance exchange membrane 30 and remain, the bubbles are released to the air reservoir 16 by tilting the entire cell culture vessel 1. After that, the ports 51 and 52 of the lower 5L of the housing are closed. If necessary, the culture medium may be perfused by constructing a flow path of the culture medium using the port 51 of the lower part of the housing 5L as an inflow path and the port 52 of the lower part of the housing 5L as an outflow path. At this stage, cells may be cultured by placing the cell culture vessel 1 in the incubator.

Next, a flow path for the nutrient medium is constructed by using the port 61 of the upper part 5U of the housing as an inflow path and the port 62 of the upper part 5U of the housing as an outflow path. Then, the port 61 of the upper portion 5U of the housing is connected to a nutrient medium supply source that supplies a fresh nutrient medium into the storage chamber 20. Further, the port 62 of the upper part 5U of the housing is connected to the tank for storing the used nutrient medium. Next, the cells are cultured by placing the cell culture vessel 1 in the incubator.

In the cell culture method of this example, the fresh nutrient medium can be continuously or sequentially supplied to the storage chamber 20 at the time of cell culture. The nutritional medium supplied to the storage chamber 20 may contain low molecular weight components such as nutritional components. Examples of low molecular weight components include nutritional components such as sugars, amino acids, lipids, peptides, and nucleic acids, salts, and buffers. These components are supplied from the storage chamber 20 to the culture chamber 10 by diffusion via the substance exchange membrane 30. In addition, waste products generated during cell culture are removed by diffusing and collecting from the culture chamber 10 to the storage chamber 20. Waste products are metabolites of cells, such as lactic acid. Therefore, in the cell culture vessel 1, cell culture can be performed even if the nutrient medium supplied to the storage chamber 20 is a medium to which no protein component is added.

In the cell culture method of this example, cells are collected after cell culture. Specifically, first, the ports 51 and 52 of the lower part 5L of the housing are opened, and the culture medium is removed by introducing a washing liquid. Next, cells are detached from the culture scaffold 80 by introducing a cell exfoliating enzyme and allowing it to act. Next, the medium for cell recovery can be flowed in from the port 51, the effluent from the port 52 can be recovered, and the cells can be recovered. As another method, a method can be used in which the cell culture vessel 1 is decomposed into 5U in the upper part of the housing, 5L in the lower part of the housing, and the like under a sterile environment, and the cells are collected from the culture chamber 10 together with the culture scaffold 80. In the case of this method, the recovery is not limited to single cells, and it is also possible to recover cells as a tissue-like structure.

[Reference Example 1]
The estimation of the serum consumption in the case of culturing hMSC using a polystyrene porous carrier (PS304012-6 manufactured by 3D Biotek) as a culture scaffold by the dialysis method applied to the present invention is shown.

When calculating the serum consumption, it is required in advance until the ^{hMSC becomes 1.9 × 10 6} cells equivalent to the confluent when hMSC is seeded on the ^{porous carrier at 8.0 × 10 4 cells.} The amount of glucose was determined experimentally to be 2.7 mg. Then, this was used as the amount of glucose required for culturing hMSC in the estimation of serum consumption. Furthermore, in the estimation of serum consumption, the nutrient medium and the culture medium are assumed to be a basal medium containing glucose and not containing serum components and a medium obtained by adding 10% by volume of serum to the basal medium, respectively. The serum concentration is assumed to be 1.0 g / L (low glucose medium) commonly used in culture. Based on these assumptions, the total amount of nutrient medium and culture medium required for hMSC culture is estimated to be 2.7 mL.

Then, as a method of culturing hMSC using the above-mentioned porous carrier as a culture scaffold by a dialysis method, the culture medium is used only as a 1 mL initial medium in which the porous carrier is completely immersed in the wells of a 12-well plate. We envision a method for maintaining a good culture environment by supplying glucose to the culture medium by contacting the culture medium with the nutrient medium via a dialysis membrane. As a result, the serum consumption when hMSC is cultured using the porous carrier as a culture scaffold is estimated to be 0.1 mL.

[Reference Example 2]
The estimation of serum consumption in the case of culturing hMSC using a polystyrene porous carrier (PS304012-6 manufactured by 3D Biotek) as a culture scaffold by a conventional medium exchange method is shown. The amount of glucose required for culturing hMSC used in this estimation of serum consumption, the nutrient medium and culture medium assumed in this estimation of serum consumption, and the glucose concentration in the nutrition medium and culture medium assumed in this estimation of serum consumption. Is the same as Reference Example 1. Therefore, as in Reference Example 1, the total amount of the nutrient medium and the culture medium required for culturing hMSC is estimated to be 2.7 mL.

Then, as a method of culturing hMSC using the above-mentioned porous carrier as a culture scaffold by a medium exchange method, the culture medium is used as a 1 mL medium in which the porous carrier can be completely immersed in a well of a 12-well plate. We envision a method for maintaining a good culture environment by periodically replacing the entire amount of the medium with an unused culture medium. As a result, the serum volume when hMSC is cultured using the porous carrier as a culture scaffold is estimated to be 0.3 mL. The minimum amount of serum consumption when hMSC is cultured using the porous carrier as a culture scaffold by the medium exchange method can be estimated to be 0.27 mL.

The present invention is not limited to the above-described embodiment and the above-mentioned embodiment, but has substantially the same configuration as the technical idea described in the claims of the present invention, and exhibits the same function and effect. Anything is included in the technical scope of the invention and includes various modifications. For example, the above-described embodiment and the above-mentioned embodiment have been described in detail in order to explain the present invention in an easy-to-understand manner, and are not necessarily limited to those having all the described configurations. Further, it is possible to replace a part of the configuration of one embodiment and embodiment with the configuration of another embodiment and embodiment, and the configuration of one embodiment and example can be replaced with the configuration of another embodiment and embodiment. It is also possible to add configurations. Further, it is possible to add / delete / replace other configurations with respect to a part of the configurations of each embodiment and each embodiment.

1 Cell culture container 5 Membrane 5U Membrane upper part 5Ua Opening 5Ub Opening 5L Membrane lower part 5La Opening 5Lb Opening 10 Culture room 16 Air storage 20 Storage room 21 First storage room 22 Second storage room 24 Partition plate 30 Material exchange membrane 31 1st material exchange membrane 32 2nd gas exchange membrane 40 Gas exchange membrane 41 1st gas exchange membrane 42 2nd gas exchange membrane 51, 52 Port 53 Air bleeding port 61, 62 Port 63 Air bleeding port 80 Culture scaffold C cells

Claims (14)

A culture room for culturing cells and
A storage chamber that stores fluid and
A substance exchange membrane that partitions the culture chamber and the storage chamber and selectively permeates a predetermined substance without permeating the cells.
A gas exchange membrane that separates the culture chamber or the storage chamber from the outside,
Equipped with
A cell culture vessel, wherein each of the culture chamber and the storage chamber has an opening through which a fluid can flow in and out. The cell culture vessel according to claim 1, wherein the fractional molecular weight of the substance exchange membrane is in the range of 500 or more and 60,000 or less. The cell culture vessel according to claim 2, wherein the fractional molecular weight of the substance exchange membrane is in the range of 1000 or more and 15000 or less. Any of claims 1 to 3, wherein the gas exchange film contains any one of silicone, polytrimethylsilylpropine, a fluoroacrylic resin, a fluororesin, polymethylpentene, and natural rubber as a main component. The cell culture container according to item 1. A cell culture method for culturing the cells in the culture chamber using the cell culture vessel according to any one of claims 1 to 4, wherein the components are added to the culture chamber and the storage chamber through the opening. Alternatively, a cell culture method comprising supplying a medium having a different component concentration. The cell culture method according to claim 5, wherein a culture medium and a nutrient medium containing a physiologically active substance are supplied to the culture chamber and the storage chamber, respectively. The cell culture method according to claim 6, wherein the physiologically active substance is a growth factor. The invention according to any one of claims 5 to 7, wherein a culture scaffold, which is a structure capable of adhering or embedding the cells, is introduced into the culture chamber, and the cells are cultured in the culture scaffold. Cell culture method. The medium is continuously or sequentially supplied to the storage chamber through the opening, and the medium is continuously or sequentially discharged from the storage chamber through the opening, and the medium is discharged in the culture chamber. The cell culture method according to any one of claims 5 to 8, wherein the cells are cultured. The ninth aspect of claim 9, wherein the cells are cultured in the culture chamber in a state where a sampling port or a component analysis sensor is installed in the opening of the storage chamber or a flow path connected to the opening. Cell culture method. Using the sampling port or the component analysis sensor, glucose, lactic acid, ammonia, glutamine, glutamic acid, oxygen, and carbon dioxide contained in the medium (the carbonic acid contains carbonic acid, carbonate ion, or hydrogen carbonate ion). The cell culture method according to claim 10, wherein the cells are cultured in the culture chamber while continuously or sequentially measuring the concentration of at least one of the components of the cell. A method for evaluating a cell growth state, which evaluates the growth state of the cells in the process of culturing the cells by the cell culture method according to any one of claims 9 to 11.
The difference in glucose concentration between the medium supplied to the storage chamber and the medium discharged from the storage chamber is measured, the glucose consumption rate is calculated from the difference in glucose concentration, and the glucose consumption rate is used in the culture chamber. A method for evaluating a cell growth state, which comprises estimating the number of cells. Further, the lactic acid concentration of the medium discharged from the storage chamber is measured, the lactic acid production rate is calculated from the lactic acid concentration, and the metabolic behavior of cells in the culture chamber is estimated from the glucose consumption rate and the lactic acid production rate. 12. The method for evaluating a cell growth state according to claim 12. A method of evaluating the growth state of the cells using the method for evaluating the growth state of the cells according to claim 12 or 13, and supplying the medium to the storage chamber or the culture chamber based on the evaluation result of the growth state of the cells. The cell culture method according to any one of claims 9 to 11, wherein the cell culture method is characterized by controlling.

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